This project tests the hypothesis that cortical connectivity and function are defined by the clonal relationships of neurons. This hypothesis is based on 2 main observations. The first observation is that the functional responses of neurons are often identical within a given cortical column. The second observation is that neurons produced from a single progenitor migrate radially outward to form what is called the ontogenetic column. Perhaps preferential connectivity within these clonaly related neurons gives rise to the functional column. A recent study has supported this hypothesis in showing that neurons arising from the same radial glial progenitor, sister neurons, have a higher degree of connectivity than their more distantly related neighbors. Although an intriguing finding, sister neurons can only account for a small fraction of cortical connections. Additionally, it is unknown if the preferential connectivity found among sister cells results in a similar functional response. What is needed to test this hypothesis is to establish the extent to which cortical connectivity and function are dependent upon clonal relationships. Since every generation included in the clonal ensemble potentially doubles the number of cells involved, an ensemble encompassing only a few generations could include hundreds of neurons. Indeed, if mother-daughter pairs and clonal cousins share the same degree of connectivity as sister neurons, clonal relationships could account for thousands of cortical connections within a column. This project will label clonally related neurons by injecting replication defective retroviruses in embryonic mice. These viruses will transduce the expression of fluorescent tags, which can be distinguished on the basis of both color and cellular localization. These unique, heritable tags will identify all subsequent generations of neurons from a given progenitor. The number of generations included in the ontogenetic column will be increased by injecting the retrovirus earlier in development. Aim 1 will test whether mother-daughter pairs and clonal cousins show enhanced connectivity in the adult mouse. To complete this aim, whole-cell slice recordings will be made on neurons of matched color (i.e., clonally related) and mismatched color to determine their connectivity. Aim 2 will test the functional significance of clonal connections by determining whether clonally related neurons have similar functional responses in vivo. These results will provide a platform on which to study the mechanism of establishing cortical connectivity as well as the effect of manipulations on the ontogenetic column. PUBLIC HEALTH RELEVANCE: The understanding and treatment of neural pathology is obfuscated by the immense complexity of the brain. The goal of this project is to determine the extent to which repeated patterns of clonally related cells define cortical connectivity and function. This information could dramatically simplify our understanding of the brain and provide a conceptual framework to better understand and treat disease.